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Previous studies reported the expression of toll-like receptors (TLRs), merely TLR2 and TLR4, and complement fragments (C3a, C5b9) in vitreoretinal disorders. Other than pathogens, TLRs can recognize endogenous products of tissue remodeling as damage-associated molecular pattern (DAMPs). The aim of this study was to confirm the expression of TLR2 and TLR4 in the fibrocellular membranes and vitreal fluids (soluble TLRs) of patients suffering of epiretinal membranes (ERMs) and assess their association with disease severity, complement fragments and inflammatory profiles. Twenty (n = 20) ERMs and twelve (n = 12) vitreous samples were collected at the time of the vitrectomy. Different severity-staged ERMs were processed for: immunolocalization (IF), transcriptomic (RT-PCR) and proteomics (ELISA, IP/WB, Protein Chip Array) analysis. The investigation of targets included TLR2, TLR4, C3a, C5b9, a few selected inflammatory biomarkers (Eotaxin-2, Rantes, Vascular Endothelial Growth Factor (VEGFA), Vascular Endothelial Growth Factor receptor (VEGFR2), Interferon-γ (IFNγ), Interleukin (IL1β, IL12p40/p70)) and a restricted panel of matrix enzymes (Matrix metalloproteinases (MMPs)/Tissue Inhibitor of Metallo-Proteinases (TIMPs)). A reduced cellularity was observed as function of ERM severity. TLR2, TLR4 and myD88 transcripts/proteins were detected in membranes and decreased upon disease severity. The levels of soluble TLR2 and TLR4, as well as C3a, C5b9, Eotaxin-2, Rantes, VEGFA, VEGFR2, IFNγ, IL1β, IL12p40/p70, MMP7 and TIMP2 levels were changed in vitreal samples. Significant correlations were observed between TLRs and complement fragments and between TLRs and some inflammatory mediators. Our findings pointed at TLR2 and TLR4 over-expression at early stages of ERM formation, suggesting the participation of the local immune response in the severity of disease. These activations at the early-stage of ERM formation suggest a potential persistence of innate immune response in the early phases of fibrocellular membrane formation.

Purpose: The cataract surgery dissatisfaction rate is 20% to 35% due to ocular surface discomfort. We investigate the ocular surface discomfort after surgical failure as a consequence of age-related parainflammation. We also aim to prevent it by immune-modulating prophylactic management., Methods: Monocentric clinical trial realized in a teaching hospital. Prospective, randomized, open-label, unmasked clinical trial. One hundred patients diagnosed with cataracts underwent phacoemulsification surgery. Groups A (<65 years; n = 25) and B (>75 years; n = 25) received surgery only. Groups C and D (both >75 years and both n = 25) used cyclosporine A 0.1% cationic emulsion (CE) eye drops or CE lubricating eye drops (both twice daily), respectively, for 30 days before surgery. Patients were followed up 90 days after surgery. The primary outcome was postoperative ocular surface failure; secondary outcomes examined the influence of prophylactic cyclosporine A 0.1% CE therapy on ocular surface outcomes., Results: Group B demonstrated greater severity regarding ocular surface signs and symptoms throughout the study period, versus all other groups. Signs/symptoms were typically lower in Group A. Group C achieved significant reductions in conjunctival Symptom Assessment in Dry Eye values ( P < 0.05), conjunctival hyperemia severity ( P < 0.01), and meibomian gland dysfunction ( P < 0.001) at Day 45, versus Group B, and tear break-up time was increased ( P < 0.001). Ocular surface inflammatory marker transcription (HLADR, intercellular adhesion molecule 1 [ICAM-1], and interleukin 6 [IL-6]) was significantly downregulated in Group C, versus Group B, at 90 days ( P < 0.05)., Conclusions: Cataract surgery induced ocular surface system failure with a clinically significant persistent inflammatory status (InflammAging) in patients older than 75 years. Prophylactic cyclosporine A 0.1% CE eye drops were associated with improved ocular surface homeostasis and reductions in inflammatory markers., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: The purpose of this study was to quantify specific aqueous humor (AH) proteins in eyes affected by posterior uveal melanoma (UM)., Methods: Thirty-six eyes affected by primary UM were included. Tumor thickness and largest basal diameter were specific clinical characteristics. Tumors were staged with the American Joint Commission on Cancer Eighth Edition (AJCC) classification. During the brachytherapy (Iodine-125) surgical procedure, both the AH sample collection and the 25-gauge transscleral fine needle aspiration biopsy (FNAB) were performed. AH samples were analyzed by immunoprecipitation and SDS PAGE techniques to quantify GNAQ, BAP1, SF3B1, and EIF1AX proteins. Cytologic material underwent fluorescence in situ hybridization for chromosome 3. The AH of 36 healthy eyes was used as the control group. Cluster analysis of groups was also performed., Results: Compared with the control group, significantly higher protein levels of: GNAQ (P = 0.02), BAP1 (P = 0.01), and SF3B1 (P = 0.02) were detected in eyes with UM. Cluster analysis of UM group revealed 2 clusters, one showing higher expression of GNAQ and BAP1 protein and one of EIF1AX protein. Moreover, the 2 clusters corresponded with the chromosome 3 status of UM., Conclusions: Specific and selected proteins may be detected in the AH of eyes affected by UM. These findings confirm the possibilities provided by AH analysis in UM.

Purpose: Ocular mucous membrane pemphigoid (OcMMP) is a rare eye disease characterized by relapsing-remitting or persisting long-lasting inflammatory events associated with progressive scarring. Despite long-term immunomodulating therapy, abnormal fibrosis keeps worsening in patients with OcMMP. This study investigates the fibrotic process in patients with OcMMP, as well as the critical role of the epithelium in modulating the local fibrosis., Methods: In this prospective, observational pilot study, patients affected by long-lasting OcMMP were compared with age- and gender-matched healthy controls. Clinical grading was assessed, and conjunctival biopsy and impression cytology were performed. Conjunctival samples were used for quantifying the expression of transcripts regulating the inflammatory and fibrogenic processes., Results: Ocular surface clinical and functional markers worsened in patients with OcMMP with fibrotic disease progression. In more advanced disease stages, both impression cytologies and conjunctival biopsies revealed increased tissue remodeling and profibrotic markers (α-SMA and TGF-β), and decreased levels of inflammatory markers (I-CAM1, IL-10, and IL-17). Increased epithelial expression of profibrotic markers and histological changes were detected., Conclusions: Chronic OcMMP is characterized by a progressive, aberrant self-sustaining fibrotic process that worsens clinical signs and symptoms. Conjunctival epithelial cells may transdifferentiate into myofibroblast-like phenotypes when chronically exposed to high levels of inflammation, as in the case of OcMMP. Tissue remodeling markers in OcMMP could be used as early diagnostic, prognostic, and therapeutic biomarkers, harvested in a non-invasive and painless procedure such as impression cytologies.

Purpose: The study aims to evaluate the usefulness of lutein/trypan blue vital dye for the staining of corneal tissues and endothelium-Descemet membrane (EDM) for Descemet membrane endothelial keratoplasty (DMEK)., Methods: Sixteen human corneal tissues (Eye Bank, Rome, Italy) were used. Corneal endothelium was tested at 25 s (T0), 1 min (T1), 2 min (T2), and 4 min (T4) from dye addition. Staining intensity and cell counting were compared. Stripped EDM was analyzed for selected apoptotic (AP, caspases, BCL2, BAX) and differentiation (VEGF-A, TGF-β1RI, SMAD3/7, SMA) targets and changes in target expression. Protein extracts were analyzed through SDS-PAGE/IB., Results: Although trypan blue staining produced the same color intensity of lutein/trypan blue dye in half the time, lutein/trypan blue reached a good and adequate color intensity at T4, which persisted even on excised and washed EDM grafts. Lutein/trypan blue-stained EDM showed a reduced number of blue-stained cells and AP immunoreactivity was significantly reduced in the same samples. An increased BCL2 transcript and a reduced BAX transcript were detected in lutein/trypan blue-stained EDM. No significant changes were observed for the main effector caspases (3/9) upon both treatments and the target genes representative of endothelial cell trans-differentiation (TGF-β1RI, SMAD3/7, SMA). A trend in vascular endothelial growth factor (VEGF-A) regulation was observed in lutein/trypan blue-treated EDM grafts., Conclusion: Obtained results suggest that lutein/trypan blue dye deserves attention in the DMEK field and support the potential routine use of this dye as a valid alternative to trypan blue for all procedures devoted to the assessment of endothelial cell viability and visualization of EDM graft before DMEK grafting., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The neurosensory retina is an outgrowth of the Central Nervous System (CNS), and the eye is considered "a window to the brain." Reelin glycoprotein is directly involved in neurodevelopment, in synaptic plasticity, learning and memory. Consequently, abnormal Reelin signaling has been associated with brain neurodegeneration but its contributing role in ocular degeneration is still poorly explored. To this aim, experimental procedures were assayed on vitreous or retinas obtained from Reeler mice (knockout for Reelin protein) at different postnatal days (p) p14, p21 and p28. At p28, a significant increase in the expression of Amyloid Precursor Protein (APP) and its amyloidogenic peptide (Aβ1-42 along with truncated tau fragment (i.e., NH 2 htau)- three pathological hallmarks of Alzheimer's disease (AD)-were found in Reeler mice when compared to their age-matched wild-type controls. Likewise, several inflammatory mediators, such as Interleukins, or crucial biomarkers of oxidative stress were also found to be upregulated in Reeler mice by using different techniques such as ELLA assay, microchip array or real-time PCR. Taken together, these findings suggest that a dysfunctional Reelin signaling enables the expression of key pathological features which are classically associated with AD neurodegenerative processes. Thus, this work suggests that Reeler mouse might be a suitable animal model to study not only the pathophysiology of developmental processes but also several neurodegenerative diseases, such as AD and Age-related Macular Degeneration (AMD), characterized by accumulation of APP and/or Aβ1-42, NH 2 htau and inflammatory markers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Balzamino, Esposito, Marino, Calissano, Latina, Amadoro, Keller, Cacciamani and Micera.)

Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m 2 ), either alone or supplemented with NGF (1-10-100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific "inflammatory to remodeling" pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling ( DNMT3a , HDAC1, NRF2 and KEAP1 )) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkA NGFR /p75 NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 transcripts, while it significantly upregulated NRF2 at lowest NGF doses and did not change KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkA NGFR /p75 NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

Aim: To develop an experimental model of endogenous nerve growth factor (NGF) deprivation by retrobulbar administration of purified neutralizing anti-NGF antibodies in young Sprague-Dawley rats and provide further information on NGF expression in the retina and cornea., Methods: Sixty old pathogen-free Sprague Dawley rats (p14, post-natal days) were treated with repeated retrobulbar injections of neutralizing anti-NGF (2 µL, 100 µg/mL, every 3d). After 2wk (p28), retinal and corneal tissues were investigated for morphological, biochemical, and molecular expression of trkA NGFR by using Western blotting or immunofluorescence. Rhodopsin as well as protein profile expression were also investigated., Results: Chronic retrobulbar neutralizing anti-NGF antibodies changed the distribution of trkA NGFR immunoreactivity at retinal level, while no changes were detected for global trkA NGFR protein expression. By contrary, the treatment resulted in the increase of corneal trkA NGFR expression. Retinal tissues showed a decreased rhodopsin expression as well as reduced number of both rhodopsin expressing and total retinal cells, as observed after single cell extraction. A decreased expression of ICAM-1, IL-17 and IL-13 as well as an increased expression of IL-21 typified retinal extracts. No significant changes were observed for corneal tissues., Conclusion: The reduced availability of endogenous NGF, as produced by chronic retrobulbar anti-NGF administration, produce a quick response from retinal tissues, with respect to corneal ones, suggesting the presence of early compensatory mechanisms to protect retinal networking., (International Journal of Ophthalmology Press.)

We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines' signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1-100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors' activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

Nerve growth factor (NGF) plays a crucial role in retinal disorders, as suggested by in vitro/in vivo models. The major effect embraces the neuroprotective activity on retinal ganglion cells (RGCs) undergoing degeneration, as observed in experimental diabetic retinopathy, age-related and diabetic macular degeneration, and some vitreoretinal diseases. Focused experiments suggested that locally applied NGF (intravitreal delivery) not only allowed the counteraction of RGC degeneration but also provided data for a whole retina restoration. The currently available retinal microsurgery allows the collection of human aqueous and more interesting vitreous (vitreal reflux) humors. The recent biomolecular analysis highlights the possibility to identify disease-associated biomarkers and allow the monitoring of retinal impairments with sustain to the retinal imaging. Coupled to other soluble mediators, NGF has been quantified in aqueous (slightly expressed) from diabetic retinopathy-suffering patients (cataract surgery) and vitreal reflux (significantly impaired) of diabetic macular degeneration-suffering patients (intravitreal surgery). Although the reasons of these NGF impairments are not fully comprehended, some retinal cells (glial cells, bipolar neurons, and RGCs) have been recognized partially responsible for these local changes.Taken together, the recent progress in the ocular microsurgeries might be associated with sampling of small amount of ocular humors, allowing the collection of biochemical information about diseased retina and the monitoring of treatment. The chance to detect NGF and likewise other neuroprotective or pro-/anti-inflammatory factors in these fluids would open to the possibility to identify biomarkers of early diagnosis or monitoring of retinal disease evolution/therapy (precision medicine)., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Purpose: To investigate osteopontin (OPN) expression in vitreous and in related idiopathic epiretinal membranes (ERMs), with respect to VEGF-A, IL8, MIP1α, IL6, and IL33, and correlate OPN expression with disease staging., Methods: Fifteen (15) vitreous and allied ERMs were collected at the time of therapeutic vitreoretinal surgery. Additional 5 vitreous and 10 ERMs (historical collection) were used. Biochemical and molecular analysis of OPN was performed in clear vitreous, vitreal pelleted cells, and ERMs. Double-immunofluorescence analysis (OPN - GFAP and OPN - αSMA) was performed on paraffin and whole-mounted ERMs. Vitreal OPN levels were correlated to those of VEGF-A, IL8, MIP1α, IL6, and IL33., Results: High OPN levels were observed in vitreal samples, and OPN transcripts were amplified in vitreal cells and related ERMs. OPN immunoreactivity was found in ERMs, mainly in GFAP-bearing (Muller cells) and to a less extend in αSMA-expressing (myofibroblasts) cells. OPN levels were highest at early stages of ERM formation and positively correlated to VEGF-A and MIP1α., Conclusions: High OPN levels in vitreous, OPN transcripts in vitreal cells/ERMs, OPN immunoreactivity in activated Müller cells and contractile myofibroblasts, as well as the correlation with VEGF-A and MIP1α fulfill the potential involvement of OPN in both inflammation and tissue remodeling that takes part in vitreoretinal interface disorders. The highest OPN levels at early stages of ERM formation would prospect OPN as a potential biomarker for disease severity.

Purpose: Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression., Methods: Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA., Results: Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05)., Conclusions: The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.

Purpose: To evaluate the changes in activity of biomarkers of Mu[Combining Diaeresis]ller cells (MC) in aqueous humor of patients with diabetic macular edema after subthreshold micropulse laser, over 1 year., Methods: Patients with untreated diabetic macular edema and central retinal thickness ≤ 400 μm were enrolled. Best-corrected visual acuity, full ophthalmic examination, and optical coherence tomography were performed. Subthreshold micropulse laser was applied every 3 months. Glial fibrillary acidic protein and inwardly rectifying potassium channel (Kir 4.1), MC activity markers, and vascular endothelial growth factor were quantified in the aqueous humor collected at baseline and at 1, 3, and 12 months after laser. Changes in the macular thickness and inner nuclear layer thickness, where MC bodies are located, were measured., Results: Ten eyes of 10 patients were included. Best-corrected visual acuity improved at 3 months (P = 0.047) and remained stable. Inner nuclear layer thickness significantly reduced at 12 months (P = 0.012). Glial fibrillary acidic protein, Kir 4.1, and vascular endothelial growth factor decreased at 1 and/or 3 and/or 12 months compared with baseline (P < 0.05)., Conclusion: Subthreshold micropulse laser improves visual function in diabetic macular edema. Kir 4.1 and glial fibrillary acidic protein decrease and inner nuclear layer thickness reduction demonstrate that subthreshold micropulse laser may restore MC function. Subthreshold micropulse laser also reduces vascular endothelial growth factor concentration. The effect of subthreshold micropulse laser in diabetic macular edema may in part be due to changes of MC metabolic activity.

Subthreshold micropulse laser (SMPL) is a tissue-sparing technique whose efficacy is demonstrated for diabetic macular edema (DME) treatment. However, its mechanism of action is poorly known. A prospective observational study was performed on naïve DME patients treated with SMPL, to evaluate the changes of aqueous humor (AH) inflammatory and vaso-active biomarkers after treatments. AH samples of eighteen DME eyes were collected before and after SMPL. Ten non-diabetic AH samples served as controls. Full ophthalmic evaluation, spectral domain optical coherence tomography (SD-OCT) and fluorescein angiography were performed in DME group. Glass chip protein array was used to quantify 58 inflammatory molecules. Central retinal thickness (CRT) and visual acuity were also monitored. Several molecules showed different concentrations in DME eyes versus controls (p value < 0.05). Fas Ligand (FasL), Macrophage Inflammatory Proteins (MIP)-1α, Regulated on Activation Normal T Cell Expressed and Secreted (RANTES) and Vascular Endothelial Growth Factor (VEGF) were increased in DME at baseline versus controls and decreased after SMPL treatments (p < 0.05). CRT reduction and visual acuity improvement were also found. Inflammatory cytokines, mainly produced by the retinal microglia, were significantly reduced after treatments, suggesting that SMPL may act by de-activating microglial cells, and reducing local inflammatory diabetes-related response.

Purpose: To determine if aqueous humour (AH) concentrations of Retinal Pigment Epithelium (RPE)'s biomarkers are modified after subthreshold micropulse laser (SMPL) treatment of diabetic macular edema (DME). Methods: Naïve DME and healthy subjects were enrolled. All DME patients received SMPL treatments (577-nm yellow light, 5% duty cycle of 0.2 s, power 250 mW), according to study protocol. AH of DME eyes was sampled at baseline and periodically after first SMPL treatment. Control eyes were sampled before cataract surgery. Pigment Epithelium Derived Factor (PEDF) and Erythropoietin (EPO) were quantified with glass-chip protein array. Results: Eighteen DME patients (central retinal thickness ≤ 400 μm on Spectral Domain Optical Coherence Tomography (SD-OCT)) and ten controls were enrolled. The main exclusion criteria were high refractive error, proliferative diabetic retinopathy, glaucoma and neurodegenerative disorders. PEDF concentration was decreased in DME patients at baseline versus controls ( P =0.012), while EPO was increased ( P =0.029). Both molecules' concentrations remained stable during follow-up after treatments, compared with DME-baseline. Conclusions : The AH concentrations of RPE biomarkers were significantly different in DME treatment-naïve eyes versus controls. The expression of PEDF and EPO remained unchanged after treatments with SMPL in DME eyes. These data are relevant for future research and applications of SMPL., (© 2019 The Author(s).)

A possible link between Nerve Growth Factor (NGF) and Reelin might take place during impaired retinal development occurring in the Reelin deficient mouse model (Reeler). To better characterize NGF and retina impairments at the Reeler retina, vitreous and retina were investigated by means of protein expression and glial cell activation. Reeler (n = 9; RELN-/-) and WT (n = 9; wild-type RELN+/+, B6C3Fe) mice were analyzed at 14, 21 and 28 postnatal days (p). Retinas and vitreous were subjected to confocal analysis and protein array, followed by conventional analysis. A significant increase of NGF, IL33 and TIMP1, a trend to a decrease of IL12 and IL6, as well as a significant decrease of NT3 were detected in Reeler vitreous, particularly at p28 (p<0.05). MIP3β mRNA was decreased while IL33mRNA was significantly upregulated in Reeler retina. Increased number of GFAP+ and Nestin+ cells as well as upregulation of Glutamine Synthetase and Nestin mRNAs were observed in Reeler retinas (p<0.05). These findings extend our previous studies on Reeler retina showing a selective Müller cell activation. NGF and IL33 release into vitreous would suggest a local activation of Müller cells, in addition to retinal ganglion and accessory cells. Overall, the data from this experimental study would strength the potential neuroprotective role played by activated Muller cells through NGF release., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To quantify inflammatory, growth/angiogenic, and tissue remodeling mediators in vitreal reflux (VR) in patients with diabetic macular edema (DME), as collected at first and third intravitreal anti-vascular endothelial growth factor (anti-VEGF, ranibizumab) injection., Methods: Thirty (30) consecutive patients (type-2 diabetes mellitus) with visual impairments due to DME and undergoing the first (untreated DME) or the third (treated DME) intravitreal injection of anti-VEGF were included in the study. At the time of surgery, patients were subjected to clinical assessment and spectral domain-optical coherence tomography (SD-OCT), including central retinal thickness (CRT), macular volume, and outer nuclear layer/retinal pigment epithelial (ONL/RPE) measurements. VR sampling was performed at the time of needle removal and subjected to customized protein-array, Western blotting (WB), Ella™ microfluidic, and/or enzyme-linked immunosorbent assay (ELISA) analysis. Biostrumental and biochemical data were collected just prior to the surgery and are representative of disease state. Clinical, biostrumental, and numerous biomarkers and cytokines were statistically compared., Results: Decreased CRT values were detected in treated DME retinas, as compared to untreated ones (p ≤ 0.05). Differences in VEGF and other mediator expressions between treated and untreated DME were detected in VR samples. Particularly, osteopontin (p ≤ 0.05), interleukin 6 (IL6) (p ≤ 0.05), and VEGF (p ≤ 0.1) values were decreased after treatment. Significant changes were validated by WB, ELISA, and Ella™ analysis., Conclusion: Overall, the biostrumental and biochemical data suggest the presence of a specific pattern of inflammation in VR after treatment. The data would suggest the presence of other mechanisms and mediators, in addition to VEGF, accountable for DME progression.

Purpose: To investigate Nerve Growth Factor (NGF) and nitric oxide synthase (iNOS) levels in tears obtained from Video Display Terminal (VDT) workers and correlate their expression with ocular signs and symptoms., Methods: A total of 120 VDT workers (62M/58F; 31-63 years old) and 40 age/sex matched no-VDT volunteers (19M/21F; 30-60 years old) were enrolled in the study. Participants completed the OSDI questionnaire and were subjected to clinical assessment of ocular surface status, including ocular symptoms and tear film parameters. NGF and iNOS levels were quantified in tear samples and their expressions correlated with OSDI, ocular symptoms and tear film parameters., Results: 59.17% of the studied population was symptomatic based on OSDI scores. Women were more commonly affected. The most frequent symptom was asthenopia and except for dryness, no differences were found between genders regarding other symptoms. A statistically significant decrease in NGF levels was found between normal and moderate (p < 0.05) and between mild and moderate (p < 0.05) OSDI grading. iNOS expression was increased in moderate OSDI grading compared to normals (p < 0.05). A negative correlation was found between NGF and respectively OSDI results, dryness and blurry vision (p < 0.05). No correlations were found among NGF, iNOS and ocular surface parameters (Schirmer, BUT, ocular surface staining)., Conclusion: Our data suggest that NGF and iNOS levels contribute to VDT ocular discomfort. Further studies are required to better understand the relationship between NGF and iNOS in VDT ocular surface.

Purpose: We characterize age-associated alterations in the expression of inflammatory mediators and tissue remodeling factors in human tears., Methods: A total of 75 consecutive volunteers (32 male/44 female; 19-93 years) underwent clinical assessment of ocular surface status, ocular surface disease index (OSDI) grading and tear sampling. The volunteers were categorized into three groups: young (18-40 years), middle-aged (41-60 years), and old (>60 years). Total protein profiles and chip-based protein array evaluations were conducted to investigate the expression of 60 potential candidates, including pro-/anti-inflammatory mediators and tissue remodeling factors. Appropriate validations were performed using conventional assays. Multiple comparisons for regression between potential candidates and age were performed, as well as statistical analyses among the three age groups. Nonpooled samples were used for quantifications., Results: Pearson analysis of chip-arrays identified 9 of 60 potential candidates. Specifically, IL-8, IL-6, and regulated on activation, normal T cell expressed and secreted (RANTES; P < 0.0083) protein as well as matrix metalloproteinase (MMP)-1, IL-3, and TNF-α (P < 0.05) correlated positively with aging. MIP-3β showed an opposite tendency. Western blot and ELISA analysis corroborated the array data. OSDI grading did not correlate with aging., Conclusions: Dynamic changes to tear protein profiles occur with aging. Our study identifies the expression of IL-8, IL-6, RANTES, MMP-1, and MIP-3β as increasing with age. These select inflammatory and matrix remodeling factors may be relevant to the development of novel diagnostic tools and therapeutics in the context of age-related ocular surface disease.

Purpose: Our previous study highlighted the potential nerve growth factor (NGF) effect on damaged photoreceptors from a rat model of spontaneous Retinitis Pigmentosa (RP). Herein, we tested the combined NGF/anti-vascular endothelial growth factor (αVEGF) effect on cultured retinal cells isolated from Royal College of Surgeons (RCS) rats receiving an intravitreal VEGF injection (iv-VEGF) to exacerbate retinal inflammation/neovascularization., Methods: RCS (n = 75) rats were equally grouped as untreated (n = 25), iv-saline (single saline intravitreal injection; n = 25) and iv-VEGF (single VEGF intravitreal injection; n = 25). Morphological and biochemical analysis or in vitro stimulations with the biomolecular investigation were carried out on explanted retinas. Isolated retinal cells were treated with NGF and αVEGF, either alone or in combination, for 6 days and cells were harvested for morphological and biomolecular analyses., Results: Infiltrating inflammatory cells were detected in iv-VEGF exposed RCS retinas, indicative of exacerbated inflammation and neovascularization. In cell cultures, NGF/αVEGF significantly increased retinal cell survival as well as rhodopsin expression and neurite outgrowth in photoreceptors. Particularly, NGF/αVEGF upregulated Bcl-2 mRNA, downregulated Bax mRNA, upregulated trkA NGFR mRNA and finally upregulated both NGF mRNA and protein., Conclusions: These data confirm and extend our previous findings on NGF-photoreceptor crosstalk, highlighting that the NGF/αVEGF combination might be an interesting approach for improving neuroprotection of RCS retinal cells and likewise photoreceptors in the presence of neovascularization. Further studies are required to translate this in vitro approach into clinical practice.

Aim: To set-up a simple technique for collecting spontaneous vitreal reflux (VR) in patients undergoing intravitreal injection. Both total protein concentration and vascular endothelial growth factor (VEGF)/Interleukin 13 (IL13) levels were used to validate the technique., Methods: Sixty consecutive patients with neovascular age-related macular degeneration (nAMD, vitreal reflux drop, VR) and 10 patients underwent vitrectomy for macular hole (whole vitreous removal) were enrolled for the study as controls. Thirty-three out of 60 patients were also subjected to tear sampling. VR sampling was performed after the intravitreal injection. Four sampling tools (10 Schirmer strips, 10 microsponges, 20 millipore filters; 20 micropipettes) were tested. Analysis of protein concentration/composition was performed between VR samples and vitreous samples to analyze the difference. The concentration of VEGF and IL 13 levels between cases and control samples were compared., Results: Millipore and micropipette techniques allowed the collection of higher protein concentrations in VR samples, comparison of both protein concentrations revealed no significant difference in the protein profile. However, the micropipette sampling was found easier to perform and did not require additional protein extraction from a solid support (membrane). Indeed, tear proteins and drug contaminants were not detected in micropipette samples. Increased VEGF levels were detected in naive VR group and to a less extend in VR group of nAMD patients undergoing intravitreal injection, with respect to the controls (macular holes). No significant differences in IL13 levels were quantified in nAMD sub-groups, as compared to naive and controls., Conclusions: Overall, we provide evidence for a safe method for sampling VR at the end of intravitreal injection. This procedure might represent an interesting approach either for the prognosis of disease or monitoring the efficacy of intravitreal therapy.

Purpose: To measure the hydraulic resistance (HR) of vitreous cutters equipped with a Regular guillotine Blade (RB) or double edge blade (DEB) at cut rates comprised between 0 and 12,000 cuts per minute (CPM) and compare it with vitreous fragment size. This was an in vitro experimental study; in vivo HR measure and vitreous sampling., Methods: HR, defined as aspiration pressure/flow rate, was measured in balanced salt solution (BSS; Alcon, Fort Worth, TX) (in vitro) and during pars plana vitrectomy of 20 consecutive patients aged 18 to 65, undergoing macular surgery. HR was recorded at increasing cut rates (500-6000 CPM for the RB and 500-12,000 CPM for the DEB; 5 mL/min flow). Vitreous samples were withdrawn and analyzed with Western and collagen type II and IX immunostaining to evaluate protein size. The main outcome measures were hydraulic resistance (mm Hg/ml/min [±SD]) and optic density for Western blot and immunostaining., Results: RB and DEB showed identical HR in BSS between 0 and 3000 CPM. Above 3000 CPM, RB HR steadily increased, and was significantly higher than DEB HR. Vitreous HR was also similar for the two blades between 0 and 1500 CPM. Above 1500 CPM, RB offered a significantly higher resistance. Western blot and immunostaining of vitreous samples did not yield a significant difference in size, regardless of blade type and cut rate., Conclusions: DEB is more efficient, offering a lower HR than RB over 1500 CPM in human vitreous. There is no viscosity reduction as a function of cut-rate between 1500 and 12,000 CPM, as HR does not vary., Translational Relevance: Future vitreous cutters will benefit of a DEB; optimal cut rate needs to be defined, and the simple increase of cut rate does not provide benefits after a certain limit to be assessed.

Purpose: Retinal glia cells (RGC) activation and release of inflammatory cytokines have been associated with development of diabetic retinopathy (DR). In this study, we evaluated by protein array the presence of aqueous humour (AH) cytokines secreted by RGC in patients with diabetes without DR and with mild DR., Methods: This is a cross-sectional, case-control study. Thirty-five subjects (diabetics and controls) underwent full ophthalmic examination and AH samples collection before cataract surgery at the Department of Ophthalmology University of Padova. AH samples were analysed for total protein concentration (Bradford method) and RGC-related inflammatory cytokines using glass chip protein arrays., Results: Twelve diabetic patients without DR, 11 diabetic patients with mild DR and 12 non-diabetic controls were included. There was no significant difference in total protein concentration among the 3 groups. Interleukin IL-1β, IL-3, interferon gamma (IFN-ɣ), (IFN-ɣ)-induced protein (IP)-10 and monocyte chemotactic protein (MCP)-2 were significantly increased in diabetics versus controls. IFN-ɣ, IL-1α, IL-3 and MCP-2 were significantly increased in diabetics without DR versus controls, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-ɣ, IL-10, IP-10, regulated and normal T cell expressed and secreted (RANTES), and soluble tumour necrosis factor receptor (sTNF-R)II were significantly increased in diabetics with mild DR versus controls. Macrophage inflammatory protein (MIP-1β), GMCSF, RANTES and sTNF-RII were significantly increased in diabetics with mild DR versus diabetics without DR (p < 0.05 at least for all)., Conclusions: Differences in expression profile of AH cytokines between diabetics, without and with mild DR, and controls have been documented. Retinal neuroinflammatory biomarkers of RGC activation evaluated in AH by protein array analysis could guide in detecting specific phenotypes with potential for personalized management., (© 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Introduction: Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis., Methods: TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies., Results: A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes., Conclusion: The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates., Funding: This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

Objective: Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC., Methods: This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated., Results: An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21, βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4, βFGF, and MMP1 were highly increased in active VKC., Conclusion: A distinct "protein tear-print" characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season.

We recently reported that increased NGF and p75(NTR) as well as decreased trkA(NGFR) characterized the Reelin-deprived (E-Reeler) retina, prospecting a potential contribution of NGF during E-Reeler retinogenesis. Herein, retinal ganglion cells (RGCs), glial cells and rod bipolar cells (RBCs) were isolated from E-Reeler retinas, and NGF, trkA(NGFR)/p75(NTR) expression and apoptosis were investigated. E-Reeler (n = 28) and E-control (n = 34) retinas were digested, and RGCs, glial cells and RBCs were isolated by the magnetic bead separation. Expression of NGF, trkA(NGFR), p75(NTR), Annexin V/PI and Bcl2/Bax was quantified by flow cytometry and validated by real-time PCR or WB. In E-Reeler retinas, NGF was significantly increased in RGCs and glial cells, p75(NTR) was increased in both RBCs and RGCs, and trkA(NGFR) was unchanged. In E-control retinas, NGF and p75(NTR) were expressed mainly in RBCs and RGCs and faintly in glial cells, while trkA(NGFR) was weakly expressed by RBCs and RGCs. In RBCs and RGCs, Annexin V expression was unchanged, while Bcl2 increased and Bax decreased selectively in E-Reeler RGCs. The data indicate that E-Reeler RBCs and RGCs overexpress NGF and p75(NTR) as a protective endogenous response to Reelin deprivation. The observation is strongly supported by the absence of apoptosis in both cell types.

Purpose: To identify early biomarkers of retinal Müller cell activation in diabetic eyes with or without clinically detectable signs of diabetic retinopathy (DR)., Methods: This study was a cross-sectional comparative case series. The aqueous humor (AH) of 34 eyes was collected in 12 healthy controls, 11 diabetic patients without DR, and 11 diabetic patients with nonproliferative DR. Full ophthalmic examination and spectral-domain optical coherence tomography were performed in all eyes. Glial fibrillary acidic protein (GFAP), aquaporin 1 (AQP1), and aquaporin 4 (AQP4) were quantified in AH samples as biomarkers of Müller cell activity by ELISA. Statistical analysis was performed with ANOVA followed by Tukey-Kramer post hoc test., Results: There was no significant difference in the age among the three groups. Mean concentration of GFAP, AQP1, and AQP4 significantly increased in diabetic eyes versus controls (P < 0.05, for each comparison). Glial fibrillary acidic protein and AQP1 showed an approximate 2-fold increase, whereas AQP4 showed an approximate 25-fold increase in diabetics with DR versus controls. In diabetics without DR, AQP4 showed an approximate 6-fold increase versus controls., Conclusions: Glial fibrillary acidic protein, AQP1, and AQP4-biomarkers of Müller cell activity-are significantly increased in human eyes with diabetes, confirming that Müller cells are precociously affected by diabetes mellitus.

Background: To assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery., Methods: Human fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST-ANOVA coupled analysis was used to compare the molecular and biochemical data., Results: The fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis., Conclusions: AM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.

Both Reelin and Nerve Growth Factor (NGF) exert crucial roles in retinal development. Retinogenesis is severely impaired in E-reeler mice, a model of Reelin deficiency showing specific Green Fluorescent Protein expression in Rod Bipolar Cells (RBCs). Since no data are available on Reelin and NGF cross-talk, NGF and trkA(NGFR)/ p75(NTR) expression was investigated in retinas from E-reeler versus control mice, by confocal microscopy, Western blotting, and real time PCR analysis. A scattered increase of NGF protein was observed in the Ganglion Cell Layer and more pronounced in the Inner Nuclear Layer (INL). A selective increase of p75(NTR) was detected in most of RBCs and in other cell subtypes of INL. On the contrary, a slight trend towards a decrease was detected for trkA(NGFR), albeit not significant. Confocal data were validated by Western blot and real time PCR. Finally, the decreased trkA(NGFR)/ p75(NTR) ratio, representative of p75(NTR) increase, significantly correlated with E-reeler versus E-control. These data indicate that NGF-trkA(NGFR)/ p75(NTR) is affected in E-reeler retina and that p75(NTR) might represent the main NGF receptor involved in the process. This first NGF-trkA(NGFR)/ p75(NTR) characterization suggests that E-reeler might be suitable for exploring Reelin-NGF cross-talk, representing an additional information source in those pathologies characterized by retinal degeneration.